Typing ofLegionella pneumophilaserogroup 1 isolates by degenerate (D-)RAPD fingerprinting

Molecularand CellularProbes(1995) 9, 405-414

Typing of Legionellapneumophilaserogroup 1 isolates by degenerate (D-)RAPD fingerprinting Sameer Sakallah, 1. W i l l i a m Pasculle, 2 Robert Lanning, 1 D a v i d M c D e v i t t 2 and David Cooper I

Department of Pathology, I Division of Molecular Diagnostics and 2Division of Clinical Microbiology, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA (Received 23 May, Accepted 14 July 1995) A new method to identify clonal strains of pathogenic bacteria has been developed recently in this laboratory. The method utilizes degenerate random amplified polymorphic DNA primers (DRAPD) to amplify random fragments in crude bacterial lysates, generating reproducible DNA banding profiles or fingerprints. We use this method to type outbreak and non-outbreak isolates of Legionellapneurnophila serogroup 1 from four hospitals near to, and affiliated with the University of Pittsburgh Medical Center. Patient isolates from a large outbreak, and nearly half of the contemporaneous environmental isolates showed the same DNA profile. Other isolates derived from non-outbreak patients showed easily distinguishable profiles. Other Legionella isolates collected between 1984 and 1994 were also analysed by this method. Our studies demonstrate that four strains were common among patient and environmental isolates at the four hospitals. These strains were also found to be different from a limited number of isolates from outside the Pittsburgh area. Because of its speed, simplicity and powerful discriminating ability, we believe that the D-RAPD approach provides epidemiologists and hospital infection control teams with a powerful tool in their efforts in analysing and terminating infection outbreaks. © 1995 Academic Press Limited

KEYWORDS: DNA fingerprinting, RAPD, degenerate RAPD primers (D-RAPD), Legionella pneumophila, legionnaire's disease, molecular epidemiology. INTRODUCTION Outbreaks of nosocomial Legionellainfections are a frequent problem in hospital environments. ~'2 Thus, it is desirable to be able to trace clonal patient and environmental isolates to their source both efficiently and reproducibly. There are over one dozen different methods designed to type strains of bacteria, and most have been successfully applied to Legionella species. These include such techniques as serotyping,3 monoclonal antibody typing/ isoenzyme analysis,s restriction enzyme analysis,6 plasmid profiling, 7 pulsed-field gel electrophoresis 8 and ribotyping. ~'s

Most of these methods generate data that are useful for epidemiological studies, but at the same time have serious limitations. Restriction enzyme analysis is expensive, time-consuming, and may fail to detect subtle genetic changes. Ribotyping is easier to interpret but is less discriminating than restriction enzyme analysis.9'1°Pulsed-field electrophoresis is more discriminating than ribotypingl~'u but is lengthy and expensive due to its requirement of special electrophoresis equipment. Plasmid profiling is limited by the fact that not all strains may harbor a plasmid.

*Author to whom correspondenceshouldbe addressedat: Departmentof Pathology,Divisionof Molecular Diagnostics,1507W Biomedical ScienceTower, Pittsburgh,PA 15261, USA. 0890-8508/951060405 + 10 $12.00/0

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© 1995 Academic PressLimited

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Many of these techniques require highly purified DNA or protein preparations which can be expensive and time consuming, particularly when a large number of specimens is to be analysed simultaneously, as is often the case in epidemiological studies. Recently, molecular epidemiological typing of Legionella pneumophila using arbitrarily primed ~'~3or repeat sequenceu PCR has been described. Random amplified polymorphic DNA (RAPD) fingerprinting methods for other pathogenic bacteria have also been developed. ~4-22 In this study, we report the utility of degenerate (D-)RAPD primers developed in this laboratory 23 in characterizing isolates of L. pneumophila serotype 1 infection at hospitals near to, and affiliated with the University of Pittsburgh Medical Center. The method is simple, fast, and reproducible and uses crude bacterial lysates as a source of template DNA. Furthermore, it is highly discriminating between strains, thus making it especially suitable for epidemiological studies. Fingerprinting results of these examinations are presented and their epidemiological implications are discussed.

MATERIALS A N D METHODS Bacteria

Seventy-three isolates derived from environmental and patient specimens which were collected between 1984 and 1994 were employed (Table 1). Twentythree patient isolates were from patients in four hospitals and two outpatient areas, all of which are located within one block from each other. Two of these, Presbyterian University Hospital (PUH) and Montefiore University Hospital (MUH) are the main teaching hospitals of the University of Pittsburgh Medical Center. Among the patient isolates were those from two nosocomial clusters at PUH, one in 1987 and another in 1992 as well as a number of sporadic isolates. One isolate (59) came from a patient with legionnaire's disease in the eastern part of Pennsylvania and had been received from the Pennsylvania Department of Health. Two isolates of Legionella sp. (49, 50) and the Burlington 1 strain of L. pneumophila (52 and its duplicate, 53) served as internal controls. Also included as controls were an isolate of L. pneumophila serotype 2 (15) and another (25) which did not belong to serogroups 1-6, but reacted to a monoclonal antibody which reacts with serogroups 1-12. Two additional isolates of Legionella species, isolated from environmental samples outside the Medical Center (49, 50) were also included as controls. Prior to being placed in our stock culture collection, all isolates had been confirmed to belong to the genus

Legionella by reaction with a commercial DNA probe (GenProbe, San Diego, CA, USA). The isolates had been stored frozen in 10% skimmed milk or on buffered charcoal yeast extract agar at 5°C. The isolates were prepared for fingerprinting analysis by subculturing on buffered charcoal yeast extract agar plates which were incubated at 37°C for 48 h. The plates were examined under a dissecting microscope to verify the purity of each culture. Next, a single colony of each isolate was picked from the plates and resuspended in 500 I~1of lysis buffer (10 mM Tris-HCI, pH 8, 1 mM EDTA, 1% Triton X-100). 23.24As a further check on the purity of the cultures, each bacterial suspension was subcultured to a blood agar plate and also examined by direct fluorescent antibody testing (SciMedx, Denville, NJ, USA) to confirm its identity. Bacterial cells were lysed to release cellular DNA by heating in lysis buffer at 95°C for 30 min. 23'24 These crude lysates were stored frozen at -20°C and used directly in PCR reactions (see below). Thirty isolates (21-50) which had been stored on slants for about 2 y~ars were found to be non-viable upon subculture. In these cases a loop-full of bacteria from the slant was s~pended in lysis buffer and processed as described ab.ove. These suspensions were also checked for contamination on blood agar and for identity by direct fluorescent antibody examination. All fingerprinting reactions were carried out in a blinded fashion. The identity of each isolate was masked throughout the analysis. Occasionally, organisms from a previous run were again included in a blind fashion to verify the reproducibility of the results (see below). Primers

A single RAPD primer (DPM34) with two-base degeneracy at positions 6 and 10 was used. The primer sequence is CGGCC[A/C]CTG[T/A]. A description of degenerate (D-) RAPD primers and their use in this application has been published elsewhere. 23

D-RAPD fingerprinting

Reaction tubes containing all reagents except template DNA and enzyme were irradiated with u.v. light for 5 min prior to amplification. PCR reactions were carried out in a final volume of 25 I~1containing buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCI, 0"001% gelatin), 200 IJ~ deoxynucleotide triphosphates, 4 mM MgCI2, 4 IJM primer(s), 5 I~1of crude bacterial lysate and 1.25

Degenerate RAPD fingerprinting of Legionella isolates

407

Table 1 Source of Legionella isolates used in this study Isolate

Year

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15-116 17 18 19 20 21 22 23 24 25:1: 26 27 28 29 30 31 32 33 34 35 36 37

1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1993 1993 1992 1992 1994 1993 1992 1992 1994 1994 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992

Source* E E E P P P P P P P E P E E P E E P P P E E E E E E E E E E E E E E E E E

PUH PUH PUH PUH PUH Hospital PUH PUH PUH Hospital OPC PUH PUH PUH PUH OPC PUH PUH Hospital Hospital PUH PUH PUH PUH MUH PUH PUH PUH PUH PUH PUH PUH PUH PUH PUH PUH PUH

B

A

B B

Isolate

Year

38 39 40 41 42 43 44 45 46 47 48 49~ 50~ 51 52 53 54 55§ 56§ 57 58 59 6011 61 62 63 64 65 66 67 68 69 70 71 72 73

1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1993 1984 1984 1987 1987 1987 1987 1987 1987 1987 1987 1992 1992 1992 1984 1990 1991 1992 1990 1992 1992 1991 1992

Source* E E E E E E E E E E E E E E C C P P P P P P P P E E E E P P E P E E P P

PUH PUH PUH PUH PUH PU H Hospital A Hospital A OPC PU H PUH Waterreservoir Waterreservoir PUH Burlington 1 strain Burlington 1 strain PUH PUH PUH PUH PUH PA Dept. Health PUH PUH MUH MUH MUH MUH MUH MUH MUH MUH MUH MUH MUH Hospital B

* Abbreviations: PUH, Presbyterian University Hospital; MUH, Montefiore University Hospital; OPC, outpatient clinics of PUH; E, environmental; P, patient; C, control. Jr L. pneumophila serotype 2.

Legionella sp. § L. micdadei. II L. pneumophila not serogroups 1-6 (see text).

U Taq DNA polymerase (Roche Molecular Systems,

Gel electrophoresis

Branchburg, N J, USA). Three separate reactions were performed in tandem using different dilutions of the crude lysates (templates). Thermal cycling was carried out in a Perkin Elmer series 9600 cycler as follows. After an initial denaturation at 94°C for 2 min, 5 cycles of 30 sec at 94°C, 90-s ramp to 30°C, 30 s at 30°C and 30s at 72°C. This was followed by 35 cycles of 30 s at 94°C, 90-s ramp to 55°C, 30 s at 55°C and 30 s at 72°C. A final 10-min extension at 72°C was performed.

PCR products from the three template dilutions were initially run side by side on agarose gels (4% Agarose 3:1, FMC, Rockland, ME, USA)to check consistency of fingerprints generated (e.g. Fig. 3). A PCR product mixture was then prepared by mixing 5 I~1 from each reaction with 3 Ill agarose gel loading dye. Ten microlitres of the mixture was electrophores.ed as de.scribed. It has been our experience23that mixing PCR products in this fashion dilutes out minor experiment-

S. Sakallah et aL

408 10 I

2O I

15 I

"'-

30

25CM II

35

40

45

1

I

I

I

50 CM I

I

I

|] 1000 bp 600 400 300

1000 bp

-

-

600 400 300

I

I

55 i

6O

65

70

I

I

I

CM I

I

1000 bp aD

A--.~ B'-~

600 400 300

Fig. 1. D-RAPD fingerprints of Legionella isolates. Crude bacterial lysates were subjected to PCR amplification as described in Materials and Methods. The combined products of reactions from three different lysate (template) dilutions were electrophoresed on 4% agarose gels as described. Lane numbers correspond with isolate numbers discussed in the text and tables. Isolate 73, which is an exact duplicate of 6, is not shown in this Figure. The three gel panels represent different time periods at which the analysis was performed. In the bottom panel, band A is present in the control and all isolate lanes, suggesting that it was derived from the water. Band B, on the other hand, is present in the control as well as some isolate lanes but at much higher intensity, suggesting that it was derived from the isolate DNA. Lane C is a water (no DNA) control. Lane M is a 100 bp size marker (Life Technologies, Gaithersburg, MD, USA).

to-experiment inconsistencies. Gels were stained with ethidium bromide and visualized under u.v. light.

profiles. A schematic representation of these profiles is shown in Fig. 2.

RESULTS

Reproducibility of the fingerprint profiles

Typing of Legionella isolates to specific fingerprint profiles

The effect of template concentration on the D-RAPD reactions has recently been studied by this group. -~3 Based on that study a strategy has been adopted where the products from reactions carried out with three different template dilutions were combined prior to electrophoresis. Figure 3 shows the banding patterns at different template dilutions of five Legionella isolates. Comparing this figure with Fig. 1 (lanes 57, 59, 60, 69 and 72), demonstrates that the apparent irregularities in the banding patterns at the different dilutions are easily smoothed out by running the combined product of the three reactions on the gel. Prior to running the combined products, the banding patterns of individual reaction products were first examined in order to detect and eliminate any significant deviation from the main banding pattern of these dilutions (not shown).

The resolution of the PCR fingerprinting reaction products by electrophoresis on agarose gels is shown in Fig. 1. In order to group these isolates under specific fingerprints, some criteria had to be defined. First, isolates were considered to be unrelated if they differed by at least two bands from another isolate. Second, bands that differed by less than 20 bp were not considered significantly different and were not used to distinguish between profiles. Using these criteria a total of 28 discrete bands could be identified, of which 12 bands were unique to specific fingerprint profiles. Each profile contained 4-7 bands ranging in size from about 225 to 2000 base pairs. As a result, the 73 isolates could be divided into 14 different

Degenerate RAPD fingerprinting of M

1

II

1II

1300 --

IV

V

VI

Legionella isolates

VII VIII

IX

X

Xl

XIl

409 XIII X l V

m

1200 -1100 -1000900-

m

m

m

800-

m m

m

m

m

m m

700-

m m

m

m

600-

m I m m

500-400-

I

m M

m m

m

m m i

m

300-

m

m

m

200-

Fig. 2. Schematic presentation of the fingerprinting profiles with the D-RAPD primer DPM34. Profiles I-XlV are shown along with the 100bp size marker (M). There are 28 discrete bands in these profiles, of which 12 are unique to individual profiles. Each profile has 4-7 bands and is different from any other profile by at least two bands. Bands that differed by less than 20 bp were not considered significantly different and were not used in this classification.

isolate 57

59

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I

I

I

I

#

60

O 1~O4 °, o, I

69

O z.OO4 o° o. I

I

I

Another check for reproducibility was performed by analysing duplicate isolates in a blind fashion. These isolates included a patient isolate (6, 73), an environmental isolate (1, 23), and a control strain (52, 53). In each case the second analysis agreed with the first.

72

O rID ¢%1 °° o. I

I

I

O tOOl °... I

I

I

I

The significance of water controls - 1000bp - 600 - 500 - 400 - 300

Fig. 3. Fingerprint analysis of Legionella isolates using different lysate dilutions. Three different dilutions of the crude lysate were used routinely in all fingerprint analyses described in this report. Prior to running the combined products of the three reactions (see text, Fig. 1), the individual reaction products were electrophoresed side by side in order to detect and eliminate any major deviation in the banding pattern due to DNA concentration. In this figure, the products of reactions using the three lysate dilutions of five isolates are shown. When these products were combined, the resulting patterns are shown in the corresponding lanes in Fig. 1.

Since Legionella sp. is an environmental organism that is often found in water supplies, it is important that water used in the reaction mixtures is included as a control in D-RAPD fingerprinting. This control has been discussed elsewhere 23and was used in every fingerprinting reaction described here. Fingerprint analysis shown in Fig. 1 was carried out at different times, represented in the three gel panels. The control (C) lanes in the top two panels do not show any bands, unlike that in the bottom panel where several bands are clearly visible in the C lane. Some of these bands are present in all lanes (e.g. fragment A) and have similar intensity which indicates that they were derived from water in reaction tubes. Other control bands (e.g. fragment B) are present in some lanes at much higher intensity and absent in others, suggesting they originated from the clinical isolate itself and unlike band B, band A was not used in typing the isolates to the profiles shown in Fig. 2.

S. Sakallah et al.

410 Table 2.

Genetic profiles of Legionella isolates: results summary

Profile

Isolate* 1984

I

1987

1990

1991

P

1992

1993

1994

(6, 73)

E II

III IV

V Vl

P E

--

58, 61

66

--

--

65

--

--

21, 24, 27, 32-34, 36-40, 42, 43, 46-48, 62-64, 68, 70, 71

11, 16

P E

(15]

P

54

10

E P E P

--

44, 45

E

67

28 4, 5, 7-9, 18

--

(1, 23), 2, 3, 13, 14, 17, 22,

12 51

19, 20

26, 29-31, 35, 41 VII

P E

VIII

P

IX X

C P

[50]

E

[25], [49] (52, 53)'1" 59

E Xl

P E

[55], [56]

XII

P

57

XIII

P E P E

XIV

69 72

{60}

* Viable isolates are shown in bold. () indicates duplicate runs of the same isolate. {} indicates isolates with serogroup other than serogroup 1. [] indicates belonging to Legionella sp. other than L. pneurnophila. +Control, Burlington 1 strain. Typing of L. pneumophila serogroup 1 isolates

Results of D-RAPD fingerprint analysis of the 73 Legionella isolates are summarized in Table 2. Sixtyfive L. pneumophila serogroup 1 isolates were divided into nine profiles (I, II, IV, V, Vl, IX, X, Xll and XlII). Twenty patient isolates collected over a 10 year period belonged to seven of these nine profiles (I, II, IV, Vl, X, Xll and XlII). A single environmental serogroup 1 isolate (28) and the Burlington 1 strain (52 and its duplicate 53), which is also serogroup 1, each gave a unique profile (V and IX, respectively). The remaining profiles (111,VII, VIII, Xl and XlV) exclusively contained either L. pneumophila isolates that were not serogroup 1 (15, 60) or other Legionella sp. isolates (25, 49, 50, 55 and 56). Five isolates (4, 5, 7, 8 and 9) from PUH patients involved in the 1992 cluster revealed that all belonged to profile Vl. The epidemiological details of this cluster are being reported elsewhere (D. Veloshin, S. Sakallah, W. Pasculle, S. Krystofiak and E. J. Wing. Nosocomial legionnaire's disease: an explosive outbreak

following interruption of hyperchlorination. Manuscript in preparation). Another PUH patient (18) with community acquired infection in December 1992 also fell in this profile. Thirteen of the 29 (46%) of the environmental isolates collected from the PUH water distribution system during the investigation of the 1992 cluster also belonged to profile Vl. The remaining 16 isolates were placed in profile II. The final isolate from the 1992 cluster (10) was from a patient which had been admitted to PUH from Hospital A with pneumonia symptoms. This isolate was found to be different from the other patients involved in the 1992 cluster and was placed in profile IV. Two environmental isolates collected from the potable water supply of Hospital A in 1992 (44 and 45) were also found to belong to profile IV. It was, however, similar to a single PUH patient isolate (54) from 1987. A sporadic isolate of L. pneumophila from a PUH patient in 1993 was also found to belong to profile Vl. It could not be determined with certainty whether this infection was nosocomial or community-

Degenerate RAPD fingerprinting of

acquired. During 1993, L. pneumophila serogroup 1 was isolated only three times from the potable water supply. An isolate from a water fountain (51) which was not related temporally or spatially to the solitary patient above was also found to belong to profile Vl. Two environmental isolates from 1993 (11, 16) and one from 1992 (46) came from outpatient areas all belonged to profile II. Among the isolates described in this report were five (54, 57, 58, 60 and 61) from a cluster of nosocomial infections of unknown origin which occurred at PUH in over 8 months in 1987. Only isolate 60 was of unknown serogroup, the rest were all serogroup 1. These isolates were placed in four separate profiles (11, IV, XII and XlV) suggesting that the 1987 cluster did not result from a common source. Two of these isolates (58, 61) however belonged to profile II which was the most prevalent profile in the PUH water distribution system during the 1992 cluster but not the profile responsible for the 1992 cases. There were no environmental isolates available for testing from 1987 because the hospital had installed a water chlorination system and we could not find Legionellae in the potable water during the cluster. Three isolates (6, 19 and 20) came from patients in Hospital B. One of these isolates (6) came from a patient who had been transferred to Hospital B in 1992 from another hospital in New York state. This isolate belonged to profile I and was unique from all other isolates described here. The other two isolates (19, 20) were isolated within a month of each other 1994 in 1994 from two patients and were thought to be nosocomial in origin. These two isolates belonged to profile II, the same profile which was prevalent in PUH 2 years earlier. Four sporadic patient isolates (66, 67, 69 and 72) from MUH in 1990 and 1991 could be placed in four separate groups. Interestingly, one isolate each belonged to profiles II (66) and Vl (67). Seven of eight MUH environmental isolates were found to belong to profile II including one from 1984 (65) and six (62, 63, 64, 68, 70 and 71 ) from 1992. None of the MUH environmental isolates could be placed in profile Vl. A single environmental isolate from 1992 (25) which is not serogroup 1 belonged to profile VIII. Finally, all isolates which were expected to be unique indeed were placed into five separate profiles. The two isolates of L. micdadei (55, 56) which were both isolated in 1987 from PUH patients were found to belong to profile XI which contained no isolates of L. pneumophila. A single patient isolate of L. pneumophila serogroup 2 generated the unique profile III. The Burlington 1 strain isolate (52 and its duplicate 53) also formed the unique profile IX which is distinct from all other L. pneumophila serogroup 1 isolates.

Legionella isolates

411

Isolates of unidentified Legionella species (25, 49 and 50) were placed in profiles VIII and IX. A single L. pneumophila isolate of unknown serogroup (60) was also placed in the unique profile XlV.

DISCUSSION Epidemiological typing of isolates of Legionella isolates is an invaluable adjunct to the investigation of both community acquired and nosocomial legionnaire's disease. PCR-based molecular typing methods have the advantage over other more traditional methods in that they are relatively simple, fast and accurate. Other typing methods, especially those using restriction enzymes, may require extensive purification of the genetic material to remove contaminants and enzyme inhibitors. One PCR-based method is DNA fingerprinting with random primers (RAPD) in which a short primer of arbitrary nucleotide sequence is used in PCR reactions to amplify polymorphic sequences under initial low stringency conditions. These conditions allow the primer to anneal to the template DNA at many sites with reproducible but less than perfect matches. The PCR product is a ladder of amplified fragments that are unique to the organism in question. RAPD fingerprinting has been used recently to type a wide range of bacteria 2s such as Klebsiella, 26 Mycobacteriay '28 Streptococci, 29 Neisseria 3° and Listeria. 31 A variation of RAPD, arbitrarily-primer (AP-)PCR, has been used to type Leptospira, 32 Acinetobacter s~ and Legionella. 1'13 Successful typing with a given random primer relies primarily on trial and error since the theoretical considerations in primer design and experimental conditions often fail to produce the expected product? 4 The use of a single degenerate primer 23facilitated the generation of multiple bands within each isolate. We successfully used this technique with crude bacterial lysates from viable as well as non-viable organisms. Results from this laboratory showed no evidence of significant changes in the banding profiles due to loss of viability (not shown). Our typing system appears to be as discriminatory as systems based on the use of restriction enzymes since it recognized 4-7 unique sequences in each isolate. Of interest is that no single band was present in all isolates examined. Finally, the uniquensss of the genetic profiles presented here was confirmed with another D-RAPD primer as well as a non-degenerate primer (not shown). Regardless of the random primer used, 14 unique profiles Were obtained, and the same isolates could be grouped under these profiles as wffh DPM34. , Analysis of the strains from our stock culture collection has provided some interesting insights into

412

S. Sakallah et al.

nosocomial and community-acquired legionnaire's disease in several Pittsburgh hospitals. In 1992, following a failure of the chlorination system, the PUH potable water supply rapidly became colonized by Legionella, and six patients developed nosocomial infection over a 2 week period. All patients involved in this cluster were infected with organisms of the same D-RAPD profile. Approximately half of the isolates recovered from the potable water supply at that time also belonged to this profile suggesting that the PUH outbreak was indeed related to the recolonization of the plumbing system. The D-RAPD typing system also enabled us to identify one patient admitted to PUH during this cluster who had nosocomial legionnaire's disease acquired at another hospital. Because L. pneumophila was not isolated from this patient until 5 days after admission to PUH, it was not certain during the outbreak whether this patient had acquired the disease in PUH or in Hospital A. D-RAPD analysis clearly identified this isolate as being different from the others and identified previously unsuspected nosocomial infection in Hospital A. Only one case of legionnaire's disease has been previously recognized in Hospital A. Because of the low prevalence of nosocomial legionnaire's disease, no water treatment programme was in operation. We found organisms matching the D-RAPD profile of this patient's isolate in the potable water supply of Hospital A. A chlorination system has been present in the potable water distribution system of PUH since 1984. Because of this system, Legionellae have not regularly been present in the water system and few nosocomial infections have been detected. In 1987, however, we detected a curious cluster of nosocomial infections between February and September. Five patients were infected with a combination of L. pneumophila serogroup 1 (58, 61, 55 and 56), L. rnicdadei (55, 56) and another serotype of L. pneurnophila (60). During this period, Legionellae could not be found in the water distribution system and the source of these patients' infections was never established. Typing of the isolates confirmed that they are not all related to each other. Two patients (58, 61) indeed had the same D-RAPD profile, but the other three each were infected with different D-RAPD types suggesting that this cluster did not have a common origin. Legionella control efforts at MUH have involved periodic heating and flushing of the potable water distribution system. One limitation of the 'heat and flush' method is that the system often becomes recolonized during the periods between treatment. We examined isolates of L. pneurnophila serogroup 1 from four sporadic cases of legionnaire's disease at MUH in 1990 and 1991 and found each to be

unrelated to the other. Of interest however is that one of these belonged to D-RAPD group II, one of two groups which was found in the PUH water distribution system in 1992. In addition, six isolates from the MUH water supply from 1992 also belonged to group II. Another patient isolate from 1991 belonged to group Vl, the group responsible for the cluster at PUH in 1992. Three cases of legionnaire's disease have been recognized at Hospital B in recent years. An isolate from one patient who had been transferred from a New York hospital with pneumonia was different from all other serogroup 1 isolates in our collection. Isolates from two cases which occurred within a month of each other in 1994 were different from the 1992 isolate but not different from each other. These two isolates also belonged to D-RAPD group Vl which was responsible for the 1992 cluster of infections at PUH. The accuracy of our D-RAPD typing system and our conclusions is further suggested by a recent repo~ s which demonstrated that the two 1994 isolates from Hospital B had similar restriction enzyme analysis profiles and that the profile matched that of contemporaneous environmental isolates from the water distribution system of Hospital B. The discriminatory ability of the D-RAPD system is suggested by our examination of three isolates from diverse geographic locations (6, 52 and 59). All belonged to unique D-RAPD groups which contained no isolates from the Pittsburgh area. Because the genomic sites selected for amplification are random and their function is unknown, it is possible by chance that a particular primer could accidentally identify a locus associated with virulence in the organisms. In the case of the primer which we employed, this appears not to be the case since we showed that 20 patient isolates could be divided into nine separate D-RAPD groups and no band was common to all of these isolates. Our analysis of patient and environmental isolates from the Pittsburgh area covering a 10 year period has also uncovered some interesting relationships among the cases seen at four hospitals located in close proximity to each other. Most interesting is the fact that the two D-RAPD profiles (11 and Vl) clearly predominated among isolates from three of the hospitals in both patient and environmental specimens suggesting that these two clones were predominant in these hospitals. The collection of environmental isolates which we examined is based on a large number of isolates collected during an outbreak in 1992. However an environmental isolate from the same hospital (65) isolated over 10 years ago also belonged to the same profile (profile II) as about half of the 1992 environmental isolates and was associated

Degenerate RAPD fingerprinting of

with sporadic cases of legionnaire's disease in 1987 and 1990 at two of the hospitals (PUH and MUH). The other profile (profile Vl) which predominated in the PUH water supply was also associated with two cases in 1994 at Hospital B. Finally one case of nosocomial legionnaire's disease at PUH in 1987 could be shown to be caused by an organism belonging to profile IV which matched patient and environmental isolates from Hospital A in 1992. The ability of PCR to amplify minute amounts of nucleic acid can present significant problems when using this technique to type organisms. This is demonstrated in one of our runs (bottom panel of Fig. 1) in which the water (no DNA) control contained several bands. Given the ubiquitous distribution of Legionellae in aqueous systems and the random nature of the primers employed it is not surprising that extraneous DNA might be present in either the water or one of the buffer components or the polymerase. While we cannot assume that the bands present extraneous Legionella DNA, the contamination of other laboratory reagents by Legionellae in water has long been recognized. Despite rigorous control and u.v. irradiation of the reagents, some bands were still detected. These bands may be derived from the Taq DNA polymerase preparation. 36 Thus, including this control in the analysis will undoubtedly simplify the interpretation of the data and may be instrumental in eliminating suspicions of sample-to-sample contamination.

ACKNOWLEDGEMENTS

The authors like to thank Dr Robert Wadowsky for providing some of the Legionella isolates used in this study. This work has been partially funded by the Pathology Education and Research Foundation (PERF).

REFERENCES

1. Gomez-Lus, P., Fields, B. S., Benson, R. F., Martin, W. T., O'Connor, S. P. and Black, C. M. (1993). Comparison of arbitrarily primed polymerase chain reaction, ribotyping, and monoclonal antibody analysis for subtyping Legionella pneumophila serotype 1. Journal of Clinical Microbiology 31, 1940-2. 2. Edelstein, P. H. (1986). Control of Legionella in hospitals. Journal of Hospital Infection 8, 109-15. 3. Ezzeddine, H., Van Ossel, C., Delmee, M., and Wauters, G. (1989). Legionella spp. in a hospital hot water system: effect of control measures. Journal of Hospital

Infection 13, 121-31. 4. Joly, J. R., McKinney, R. M., Tobin, J. O., Bibb, W. F., Watkins, I. D. and Ramsay, D. (1986). Development of a standardized subgrouping scheme for Legionella

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